Field of Invention
The present disclosure provides compositions and methods utilizing double strand ribonucleic acid (dsRNA) to control parasitic nematodes, including Pratylenchus penetrans. More particularly, the present invention relates to several specific synthetic dsRNAs that induce RNA interference (RNAi) in the target nematodes and methods of delivering the dsRNAs to them.
Background
Root lesion nematodes are considered the third most important group among plant-parasitic nematodes (Jones et al., Mol. Plant Pathol., (2013) 14:946-61). Within this group of nematodes, Pratylenchus penetrans (Cobb, 1917) Filipjev & Schuurmans Stekhoven, 1942 is considered a cosmopolitan species worldwide, having a preferential distribution along temperate regions. This species has been recorded as being associated with more than 400 plants, and considered to be a limiting factor for the production of important agronomic [e.g. alfalfa (Medicago sativa L.), corn (Zea mays subsp. mays L.), potato (Solanum tuberosum L.)], ornamental [e.g. lily (Lilium candidum L.), roses (Rosa spp.)] and fruit plants [e.g. apple (Malus pumila Miller), cherry orchards (Prunus spp.), raspberry (Rubus spp.)] (Castillo and Vovlas, in “Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management. Nematology Monographs and Perspectives, 6” Brill Leiden-Boston, The Netherlands-USA (2007) p. 529). In some countries P. penetrans is considered as an A1 quarantine plant pest due to its potential impact on economic important crops (EPPO Global database).
Like other nematodes, the life cycle of P. penetrans is punctuated by six stages (eggs, four juvenile stages and adults). With the exception of the egg and J1 stages, all the remaining stages are motile and able to enter the roots and cause damage (Castillo & Vovlas, supra). This nematode is a migratory endoparasitic species that feeds and migrates within the root cortical tissue causing a reduction in root growth after infection, accompanied by the formation of lesions, necrotic areas, browning and cell death (Fosu-Nyarko & Jones, Ann. Rev. Phytopathol., (2016) 54:253-78). As migratory endoparasites the destruction of the root system can cause surface wounds, which allow access to a combination of other soil borne pathogens, such as fungi (Rotenberg et al., Plant Pathol., (2004) 53:294-302) and bacteria (Vrain et al., Can. J. Plant Pathol., (1987) 9:236-40), leading to a severe damage of the plant.
Silencing core genes through RNA interference (RNAi) can promote lethal or inhibitory effects (Lilley et al., Parasitol. (2012) 139:630-40; Danchin et al., PLoS Pathogens (2013) 9:e1003745), making it is a very promising tactic in the control of plant-parasitic nematodes. However, one imperative factor is the identification of plant-pathogen specific genes, or target sequences, that lack homologs in non-target organisms such as mammals, plants or beneficial insects (Danchin et al., supra).
Fatty-acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins, which have high binding affinity for fatty acids, retinol and retinoic acids, and are exclusively found within the phylum Nematoda (Kennedy et al., “The unusual lipid-binding proteins of nematodes: NPAs, nemFABPs and FARs”, In Parasitic nematodes: molecular biology, biochemistry and immunology, eds. Kennedy MW, Harnett W. CABI, Wallingford, UK. (2013) pp. 397-412). This family of proteins occurs in several isoforms of approximately 20 kDA, which can be found in varying numbers among species of the different clades of Nematoda (Garofalo et al., J. Biol. Chem., (2003) 278:8065-74; Iberkleid et al., Eur. J. Plant Pathol. (2015) 143:133-49). In the case of free-living nematodes, eight isoforms have been found within the genome of Caenorhabditis elegans (Maupas, 1900) Dougherty, 1955 (Garofalo et al., supra), whereas in Pristionchus pacificus Sommer, Carta, Kim & Sternberg, 1996, nineteen members have been identified (Dieterich et al., Nature Genetics (2008) 40:1193-98; Dillman et al., Genome Biol., (2015) 16:e200). In the case of animal- and plant-parasitic nematodes the number of effective FAR isoforms is still unknown. In Necator americanus (Stiles, 1902) Stiles, 1906 at least six FAR genes have been identified, while for a substantial portion of studied species so far, a single gene has been reported (Kennedy et al., J. Biol. Chem., (1997) 272:29442-48; Garofalo et al., 2003, supra; Tang et al., Nature Genetics (2014) 46:261-71; Iberkleid et al., 2015, supra). In plant-parasitic nematodes, a single FAR gene has been reported for both sedentary [e.g. Globodera pallida (Stone, 1973) Behrens, 1975 (Prior et al., Biochem. J. (2001) 356:387-94), Meloidogyne javanica javanica (Treub, 1885) Chitwood, 1949 (Iberkleid et al., PLoS One, (2013) 8:e64586), Heterodera avenae Wollenweber, 1924 (Le et al., 2016) and H. filipjevi (Madzhidov, 1981) Stelter, 1984 (Qiao et al., 2016)] and migratory [e.g. Aphelenchoides besseyi Christie, 1942 (Cheng et al., PLoS One, (2013) 8:e66011), Radopholus similis (Cobb, 1893) Thorne, 1949 (Zhang et al., PLoS One (2015) 10:e0118414)] species. More recently, comparative transcriptome and genomic data suggested that plant-parasitic species (e.g. Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 and Heterodera avenae) have additional members of this FAR family (Dillman et al., supra; Espada et al., Mol. Plant Pathol., (2016) 17:286-95; Qiao et al., PLoS One, (2016) 11:e0160003). In entomopathogenic nematode species of the genus Steinernema, a wide expansion of the number of genes within the FAR family have been reported, ranging between 38 to 54 gene members of this family (Dillman et al., supra).
FAR proteins seem to play a relevant role in the binding of lipids from their environment or host, as nematodes are unable to synthesize de novo fatty acids (Kennedy et al., 2013, supra). A wide range of functions has been implicated for nematode FARs, such as scavenging, transport and metabolism of hydrophobic lipophilic molecules like fatty acids, eicosanoids, retinoids, and steroids (McDermott et al., Mol. Cell. Biochem., (1999) 192:69-75); Kennedy, Biochim. Biophys. Acta, (2000) 1476:146-64). These proteins have important functions as energy sources and are used in the metabolic and developmental processes such as embryogenesis, glycoprotein synthesis, growth and cellular differentiation (McDermott et al., supra; Kennedy, 2000, supra). In addition to their role in the nematode's physiological activity, FARs have raised strong interest because of their implication in the parasitism process of both animal- and plant-parasitic nematodes (Kennedy et al., 1997, supra; Bradley et al., Trends Parasitol., (2001) 17:471-475; Prior et al., supra; Iberkleid et al., 2013, supra).
Previously we have identified in the transcriptome of P. penetrans a highly abundant transcript encoding for a fatty acid- and retinoid-binding gene (Vieira et al., PLoS One, (2015) 10:e0144674). Along the in silico analyses carried out along this study we were able to identify three different FARs members of this family to P. penetrans. We set out to confirm the expression levels of Pp-far-1 in different nematode developmental stages, and at earlier time points of nematode infection in different host plants. Pp-far-1 was then targeted using plant-mediated RNAi silencing assays to evaluate the importance of this gene during parasitism. Based on our RNAi analyses, knocking-down this gene via RNAi is an option to control this species.